One of the most serious problems of contemporary medicine, veterinary medicine, plant production and industry lies in the activity of various microorganisms, especially bacteria and fungi, that cause diseases and damage of products. The fight against bacteria and fungi remains insufficiently effective due to great variability of microorganisms that leads to the emergence of resistant forms, the inability to grow significant amounts of microbes and the absence of effective methods for choosing an antimicrobial means. For this reason during the first 3-4 days the antimicrobial preparations are used empirically. In medicine and veterinary medicine various broad-spectrum antibiotics are used in such situations, but the number of such antibiotics is limited, which leads to quick formation and propagation of resistance towards such antibiotics, resulting in complications and, at times, even death in medicine and veterinary medicine, and irreversible damage of food and industrial products.
In this regard a quick and precise assessment of sensitivity of microorganisms to antimicrobial preparations—antibiotics and antiseptics—constitutes an urgent and yet unsolved problem.
Known methods of determining the sensitivity to antimicrobial preparations comprise preliminary isolation of pure culture of a microorganism and identification of said pure culture.
However, the isolation of pure culture of microorganisms has some negative implications, most notably the loss of time (48-96 hours). Furthermore, if two or more microorganisms are present, some of them will not be isolated as a pure culture. It should also be noted that some “not-yet-cultivated” microbes cannot be isolated at all, and according to various sources such microbes can constitute up to 90-95% of all microbes.
The difficulties of dealing with such microbes consist mainly in the absence of methods for isolating, growing and identifying these microbes. The latter is due to the way the life of bacteria is organized within microbial communities (biofilms), which determines great interdependence between microorganisms. These conditions as yet cannot he recreated in a laboratory, see Lewis K., Epstein S. S. Persisters, biofilms, and the problem of culturability in incultivated microorganisms. In Series: Microbiology Monographs. Steinbuchel A. (ed.). Berlin/Heidelberg: Springer; 2009, pp. 181-194, Epstein S. S. General model of microbial uncultivability in uncultivated microorganisms. In Series: Microbiology Monographs. Steinbuchel A. (ed.), Berlin/Heidelberg: Springer; 2009, pp. 131-150.
A known method comprises applying antimicrobial substances onto strips in a certain way, see Isenberg H. D. Essential Procedures for clinical Microbiology, ASM-Press, 1998, USA, pp. 235-240. The serial dilution method comprises inoculation of a pure culture onto a special-purpose fluid medium Muller Hinton with the addition of antimicrobial substances (antibiotics) in varying concentrations, see Isenberg H. D. Essential Procedures for clinical Microbiology, ASM-Press 1998, USA, pp. 216-223. Some methods of semi-automatic determination of sensitivity to antibiotics employ detection by means of special-purpose equipment (e.g. Vitec 2 BioMerie panels). However, the method also uses preliminary isolation of pure culture of the assumed causative agent with subsequent exposure thereof in wells of the panel. Genetic methods allow detecting certain studied genes of antibiotic resistance among identified bacteria or even directly in the pathologic material. Said method is relatively quick, however it does not yet allow fully evaluating the sensitivity to antimicrobial preparations. This is due to the fact that many microorganisms have not been studied yet and therefore there are no data on their genome or the genes of antibiotic resistance thereof. Secondly, even among known and cultivable bacteria the resistance to a specific antimicrobial preparation can be encoded by completely different genes, many of which remain unstudied.
Another known method for determining the sensitivity of microorganisms to an antimicrobial substance comprises taking biological material and isolating, a pure culture of the microorganism. In order to isolate a pure culture, the biological material is inoculated in nutrient media that provide favorable conditions for the growth of a particular microorganism that presumably causes diseases or damage of products and materials. If the microorganism growth is successful, the microorganism is identified. Then the sensitivity thereof to an antimicrobial substance is determined by means of inoculation of isolated pure culture of the microorganism on Muller Hinton nutrient medium or a similar non-rich medium that does not contain the antimicrobial substance, followed by the placement of paper disks saturated with various antimicrobial substances upon the surface of the nutrient medium inoculated with microorganisms, and incubation of the microorganisms in the nutrient medium in the presence of the antimicrobial substances being investigated with subsequent assessment of the results, see Isenberg H. D. Essential Procedures for clinical Microbiology, ASM-Press, 1998, USA, 1998, pp. 208-215.
Yet another known method for determining the sensitivity of microorganisms to an antimicrobial substance (antibiotics) comprises taking biological material, cultivating the microorganisms contained therein on a nutrient medium that does not contain the antimicrobial substance, introducing the antimicrobial substance being investigated into the medium and subsequently assessing the results; the biological material is cultivated in sugar broth for at least two hours, then standard disks containing the antibiotics being investigated are introduced and the medium is further incubated for at least two hours, the result is assessed by the degree of opacity of the sample contents: the less the opacity, the higher the sensitivity of the microorganisms to the antibacterial substances, see RU 2262533C2.
The disadvantages of this method, which has been taken as a prototype of the present invention, consist in the following:                fairly low precision of the results' assessment, because the reduction of opacity of the sample contents can be caused by inhibition of growth of microbes other than the causative agents, the sensitivity of which to an antimicrobial substance is investigated;        since the biological material is for a long time (>2 hours) contained in a nutrient medium that does not include an antimicrobial substance, the selective growth of some microorganisms changes the overall composition of microorganisms as compared to the original material, which drastically distorts the results of the study;        introducing the antimicrobial substance to the nutrient medium on standard disks is reasonable only for isolated pure cultures, because the reference values of areas of inhibition of microorganism growth, which would allow evaluating their sensitivity and resistance to a particular antimicrobial substance, are defined only for pure cultures. In said method the pure culture of the microorganism is not isolated, thus making it impossible to take into account the microorganism's specifics or the maximum possible concentration of the antimicrobial substance at the place where the biological material is taken;        using a non-rich fluid nutrient medium does not enable growth of the majority of microorganisms.        
It is an object of the present invention to provide a solution for increasing the precision of determining the sensitivity of microorganisms to an antimicrobial substance.